Nandi Li, 1 Xiao Bo Feng, 2,3 Qian Tan, 1 Ping Luo, 1 Wei Jing, 1 Man Zhu, 1 Chunzi Liang, 1 Jiancheng Tu, 1,3 and Yong Ning 3. 1.

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1 Hindawi Disease Markers Volume 217, Article ID , 8 pages Research Article Identification of Circulating Long Noncoding RNA Linc152 as a Novel Biomarker for Diagnosis and Monitoring of Non-Small-Cell Lung Cancer Nandi Li, 1 Xiao Bo Feng, 2,3 Qian Tan, 1 Ping Luo, 1 Wei Jing, 1 Man Zhu, 1 Chunzi Liang, 1 Jiancheng Tu, 1,3 and Yong Ning 3 1 Department of Clinical Laboratory Medicine and Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China 2 Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China 3 School of Laboratory Medicine, Hubei University of Traditional Chinese Medicine, Wuhan, Hubei, China Correspondence should be addressed to Jiancheng Tu; jianchengtu@whu.edu.cn and Yong Ning; ningyong128@163.com Received 4 June 217; Revised 12 July 217; Accepted 27 July 217; Published 19 November 217 Academic Editor: Olav Lapaire Copyright 217 Nandi Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Long noncoding RNAs (lncrnas) have been reported to play vital roles in non-small-cell lung cancer (NSCLC). Recently, long noncoding RNA Linc152 has been reported to play important roles in various cancers. In this study, our aim was to investigate its expression pattern and clinical significance and further evaluate its diagnostic value for NSCLC. Methods. The levels of Linc152 were detected in NSCLC tissues and plasma samples by quantitative real-time PCR (qrt-pcr). Receiver operating characteristic (ROC) curves were depicted to evaluate the diagnostic value. Results. We found that Linc152 levels were upregulated in both NSCLC tissues and plasma samples. Plasma Linc152 levels were significantly lower in postoperative samples than in preoperative samples. Besides, high Linc152 expression was significantly correlated with tumor size (r =293, P =5) and tumor stage (r =324, P =11). The ROC curves indicated that plasma Linc152 has high diagnostic accuracy for NSCLC, and the area under curve (AUC) for NSCLC versus healthy was.816 (95% CI: ). Moreover, we found that the combination of Linc152 and CEA could provide a more powerful diagnosis efficiency than Linc152 or CEA alone (AUC =.881, 95% CI: ). Conclusions. Plasma Linc152 could serve as a promising biomarker for diagnosing and monitoring NSCLC. 1. Introduction Lung cancer is the most common cancer worldwide. Cancer statistics reported that China had an estimated 4292, new cancer cases and 2814, cancer deaths in 215, with lung cancer being the leading cause of cancer death [1]. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases, which includes squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and several other types [2]. Although some encouraging progresses in treatment for cancer have been achieved, the 5-year survival rate of NSCLC patients is still frustrated [3]. At present, the sensitivity of traditional biomarkers of NSCLC, such as CYFRA21, CEA, NSE, and CA19-9, is far from satisfaction. Therefore, it is urgent to find new noninvasive and sensitive biomarkers to complement and improve the current NSCLC screening methods. Tumor cells could release substantial amounts of RNA into the peripheral blood and are present at sufficient levels for quantitative analyses [4]. Circulating micrornas (mir- NAs) have been shown to be stable and could serve as reliable biomarkers for cancers [5 7]. Recently, many studies have focused on long noncoding RNAs (lncrnas) [8 1]. LncRNAs are a type of transcripts longer than 2 nucleotides in length, which are poorly conserved and not translated into proteins [11]. Multiple lines of study have demonstrated that lncrnas play a vital role in many biological processes, especially in tumor biology [12]. It has been

2 2 Disease Markers reported that circulating lncrnas could reflect the pathological and physiological change of cancer patients, which attracted much attention on identification of circulating lncrna as a biomarker for cancer patients [13, 14]. For instance, circulating lncrna HULC could serve as a novel biomarker for gastric cancer [15]. However, there are still few studies regarding circulating lncrnas for the diagnosis of NSCLC patients. Linc152, a novel lncrna located at 2p11.2, has been demonstrated to be upregulated in gastric cancer [16, 17], hepatocellular carcinoma [18, 19], clear cell renal cell carcinoma [2], colon cancer [21], and gallbladder cancer [22, 23]. Moreover, recent studies indicated that Linc152 could act as a circulating biomarker for esophageal squamous cell carcinoma and gastric cancer [24, 25]. However, the potential clinic value of Linc152 in NSCLC has not been investigated. Therefore, in this study, we aimed to study the expression pattern and clinical significance of Linc152 in NSCLC and then further investigate whether circulating Linc152 could serve as a potential biomarker for NSCLC. 2. Material and Methods 2.1. Ethics Approval. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (Ethical Approval number 21359). All the experiments were conducted according to the institutional ethical guidelines Sample Collection. 72 paired NSCLC tissues and corresponding adjacent normal tissues were collected from patients who underwent surgical resection without preoperative chemotherapy or radiotherapy from Zhongnan Hospital of Wuhan University between January 215 and July 216. All NSCLC tissues and corresponding adjacent normal tissues were stored at 8 C before use. We collected whole blood samples from 1 NSCLC patients (including 24 patients whose tissues were collected), 1 healthy controls, and 48 benign lung disease patients. Benign lung disease patients included pulmonary tuberculosis (n =14), pulmonary inflammatory pseudotumor (n =8), pulmonary benign sarcoidosis (n =12), and pneumonia (n =14). The whole blood samples were obtained from NSCLC patients before any anticancer treatment such as surgery, radiotherapy, and chemotherapy. Then, we collected 47 paired postoperative whole blood samples after approximately one month. Plasma was separated from a whole blood sample by two-step centrifugation (2g for 5 min at 4 C and 12g for 5 min at 4 C) to exclude cellular nucleic acids. All NSCLC patients were recruited based on the histopathology results. Tumor stage was determined according to the National Comprehensive Cancer Network (NCCN) criteria. We collected clinical information of participants from the clinical laboratory of Zhongnan Hospital of Wuhan University, including age, gender, pathology type, tumor size, tumor stage, lymph node metastasis, and tumor biomarkers. Table 1: Within-batch and between-batch coefficient of variation (CV) of Linc152 and GAPDH in the two groups. Group Linc152 GAPDH Within-batch CV (%) NSCLC Healthy control Between-batch CV (%) NSCLC Healthy control NSCLC: non-small-cell lung cancer RNA Extraction and Reverse Transcription. We used Trizol reagent (Invitrogen, CA, USA) to extract total RNA from tissues and used the blood sample total RNA rapid extraction kit (BioTeke, China) to extract total RNA from plasma according to manufacturer s instruction. Then, RNA was reverse transcribed to cdna by using PrimeScript RT reagent kit (Takara, China) in 2 μl solution containing 7 μl of RNA extract. The reverse transcription procedure was as follows: 42 C for 2 min, 37 C for 15 min, and 85 C for 5 sec Real-Time PCR Analysis. Real-time PCR (RT-PCR) was performed on the Bio-Rad CFX96 (Bio-Rad, CA, USA) using the SYBR Premix Ex TaqTM II real-time PCR kit (Takara, China) in a 2 μl reaction volume. The reaction mixtures were incubated at 95 C for 5 min and then 4 cycles of denaturation at 95 C for 3 sec, annealing at 63.4 C for 3 sec, and elongation at 72 C for 3 sec. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found to be stably expressed in plasma and was regarded as an ideal internal control. In this study, GAPDH was selected as an internal reference gene to normalize the results of RT-PCR. We found that GAPDH levels were stable in tissues and plasma (P =34). The sequence of primers were as follows: Linc152 (forward: 5 -GTGATGTCCCCAGTGATCCA-3 and reverse: 5 -TAT TCGAGGGATGCAGACGG-3 ); GAPDH (forward: 5 -GG TCTCCTCTGACTTCAACA-3 and reverse: 5 -GTGAGGG TCTCTCT-CTTCCT-3 ). The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the threshold in RT-PCR. The relative expression was calculated by using the 2 ΔCt, and ΔCt = Ct target gene Ct GAPDH. All samples were analyzed in duplicate with no-template controls included Statistical Analysis. All statistical analyses were performed using SPSS software package version 19. (SPSS, Chicago, IL, USA). The figures were drawn by GraphPad Prism 5. software (GraphPad Software, La Jolla, CA, USA). The paired-sample t test was used to compare differences of Linc152 expression in paired tissues and paired plasma samples before and after surgery. The independent two-tailed test was used to compare the levels of Linc152 between NSCLC plasma samples and healthy control plasma samples. The two-side chi-square test was used to analyze the association between the expression level of Linc152 and clinicopathological features. Correlations between the expression level of plasma and tissue were analyzed using

3 Disease Markers 3 Relative Linc152 expression (log) 4 5 Tumor (a) Nontumor Linc152 fold change (b) Figure 1: (a) The expression levels of Linc152 in NSCLC tissues and adjacent normal tissues, P <1; (b) Waterfall plot showed Linc152 was increased by at least twofold in 55.6% (39/72) of NSCLC tissues. The gray areas represent that linc152 was upregulated by at least twofold. Fold change was represented by 2 ΔΔCt, and ΔΔCt = [(Ct Linc152 Ct GAPDH ) of NSCLC (Ct Linc152 Ct GAPDH ) of normal]. Table 2: Correlation between clinicopathological features and Linc152 expression in NSCLC patients. Characteristics Numbers High expression n =36 Low expression n =36 Univariate analysis Multivariate analysis Gender Male Female Age (years) < Histology subtype ADC SCC Other Tumor size cm >3cm Tumor stage I/II III/IV Lymph node metastasis No Yes SCC: squamous cell carcinoma; ADC: adenocarcinoma; P <5 was considered significant. the Spearman correlation. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) were applied to evaluate the diagnostic value. P <5 was considered to be statistically significant. 3. Results 3.1. Accuracy of RT-PCR Methods. Within-batch and between-batch coefficient of variation (CV) of Ct value were used to evaluate the repeatability and precision of the RT- PCR. The within-batch CV of Linc152 and GAPDH for NSCLC patients and healthy controls were all <5%, and the between-batch CV were all <5% (Table 1) Linc152 Was Significantly Upregulated in NSCLC Tissues. Aiming to investigate the expression pattern of Linc152 in NSCLC, Linc152 expression levels were measured in 72 paired NSCLC tissues and corresponding adjacent normal tissues by RT-PCR. As shown in Figure 1(a), Linc152 expression levels were remarkably upregulated in NSCLC tissues compared to adjacent normal tissues (P <1). Besides, waterfall plot showed that Linc152 was upregulated in 7.8% (61/72) of NSCLC tissues and was increased by at least twofold in 55.6% (39/72) of NSCLC tissues (Figure 1(b)). These results indicated that Linc152 may exert an oncogenic role in NSCLC. NSCLC patients were divided into the low expression group (n =36) and high expression group (n =36) according

4 4 Disease Markers Ct value 2 Ct value h 6 h 12 h 24 h Incubation of plasma at room temperature Number of freeze-thaw cycles (a) (b) Figure 2: The stability of Linc152 plasma in harsh environments. Incubation of plasma at room temperature (a) and the freeze-thawing processes (b). to the median expression level of Linc152. The correlation between Linc152 and clinicopathological features of NSCLC patients was analyzed. The univariate analysis revealed that high Linc152 expression was significantly correlated with tumor size (P =5) and tumor stage (P =11, Table 2). However, the multivariate regression analysis showed that Linc152 expression was only significantly associated with tumor size (P =5, Table 2) Analysis of Stability of Linc152 in Plasma. To analysis the stability of Linc152 in plasma, five healthy plasma samples were divided into four parts and left under harsh conditions such as incubation at room temperature for, 6, 12, and 24 hours or repeated freeze-thaw cycles. No obvious alterations were observed in plasma Linc152 levels under both of the above conditions (P >5, Figures 2(a) and 2(b)). These results demonstrated that Linc152 remained stable in plasma and whole blood, which laid the foundation for detecting Linc152 levels in plasma Increased Linc152 in Plasma of NSCLC Patients. Previous results showed that Linc152 levels were significantly upregulated in NSCLC tissues and remained stable in whole blood and plasma. Thus, we further measured plasma Linc152 levels in 1 NSCLC patients and 1 healthy controls. As shown in Figure 3, plasma Linc152 levels were remarkably higher in NSCLC patients than in benign lung disease patients and healthy controls (P <1). However, there was no difference between benign lung disease and healthy controls (P =169). The clinical information of studied subjects was summarized in Supplementary Table 1 available online at There was a significant difference in concentrations of red blood cells (RBC) and hemoglobin (HGB) between the two groups. However, no difference was observed in age, gender, white blood cells (WBC), and platelets (PLT) between the two groups. We examined the associations of plasma Linc152 levels with NSCLC traditional tumor biomarkers. Plasma Linc152 levels had a statistical relevance with CEA (P <1, Supplementary Table 2). Relative Linc152 expression (log) Diagnostic Value of Plasma Linc152 for NSCLC. To further assess the potentiality of clinical application of plasma Linc152, ROC curves were constructed on data from 1 NSCLC patients and 1 healthy controls. The AUC of Linc152 was.816 (95% CI: , Figure 4(a)) for distinguishing NSCLC patients from healthy controls, and the optimal sensitivity and specificity were 8% and 72%, respectively (Table 3). Meanwhile, Linc152 also has a good diagnostic efficiency for differentiating NSCLC patients from benign lung disease (AUC =.742, 95% CI: , Figure 4(b)). Our results showed that Linc152 has an association with CEA; thus, we combined Linc152 with CEA in diagnosing NSCLC. The AUC of CEA was.741 (95% CI: ), which was lower than Linc152. We found that the combination of Linc152 and CEA could greatly improve the diagnostic efficiency for diagnosing NSCLC (AUC =.881, 95% CI: , Figure 4(c)). Furthermore, we further evaluated the diagnostic value of plasma Linc152 levels for early stage NSCLC patients (stage I). The AUC of Linc152 was.786 (95% CI: ), which was higher than that of CEA (AUC =.738, 95% CI: ). When we combined Linc152 and NSCLC Control Lung benign disease Figure 3: The levels of plasma Linc152 in NSCLC patients (n = 1), healthy controls (n = 1), and benign lung disease (n =48). P <5.

5 Disease Markers Sensitivity.6.4 Sensitivity Specificity Specificity (a) (b).8.8 Sensitivity.6.4 Sensitivity Specificity 1 Specificity Linc152 CEA Linc152 + CEA (c) Linc152 CEA Linc152 + CEA (d) Figure 4: (a-b) ROC curve for distinguishing NSCLC patients from healthy controls (a) or benign lung disease patients (b). (c-d) ROC curves to compare the diagnostic performance of Linc152, CEA, and a combination of Linc152 and CEA to discriminate all NSCLC patients (c) or early stage NSCLC patients (d) from healthy controls. Table 3: Use of Linc152 and CEA levels to distinguish NSCLC patients from healthy control. SEN SPE AC PPV NPV Linc152 8% 72.% 76.% 74.1% 78.3% CEA 44.% 86.% 65.% 75.9% 6.6% Linc152 + CEA 76.% 83.% 79.5% 81.7% 77.6% SEN: sensitivity; SPE: specificity; AC: diagnostic accuracy; PPV: positive predictive value; NPV: negative predictive value. CEA to diagnose stage I NSCLC patients, the diagnostic value was significantly improved (AUC =.844, 95% CI: , Figure 4(d)) Evaluation of the Use of Linc152 for Monitoring Tumor Dynamics in NSCLC Patients. We carried out two experiments to investigate whether plasma Linc152 levels could monitor NSCLC dynamics. Firstly, RT-PCR was used to measure the expression in 24 paired NSCLC tumor tissues and plasma samples which were from the same individuals. The correlation of Linc152 levels between the two groups was analyzed. Linc152 levels has a significant correlation between tissue and plasma (r =49, P =15, Figure 5(a)). Then, Linc152 levels were analyzed in 47 paired preand postoperative plasma samples (after one month) from NSCLC patients. The results showed that plasma Linc152 levels were significantly lower in postoperative samples than in preoperative samples (P <1, Figure 5(b)). However, compared to NSCLC patients without recurrence, plasma Linc152 levels of patients with recurrence were sharply increased (P <5, Figure 5(c)). Based on the above results, we could presume that plasma Linc152 levels might reflect tumor dynamics. 4. Discussion With the development of microarray and massively parallel sequencing technology, accumulating studies indicated lncrnas could serve as oncogenes or tumor suppressors during carcinogenesis [26]. Recently, several circulating lncrnas have been characterized as potential tumor markers for cancers. However, there are still few studies reported circulating lncrnas in the diagnosis of NSCLC patients. In this study, we set out to explore the expression pattern and clinical significance of Linc152 in NSCLC and then further investigate whether Linc152 could serve as a potential biomarker for NSCLC.

6 6 Disease Markers Linc152 in plasma (log) r =.568 p = 3 Linc152 in tissue (log).5 Relative Linc152 expression (log) 4 5 Preoperation Postoperation Relative Linc152 expression (log) 4 5 Preoperation p > 5 Postoperation Recurrence (a) (b) (c) Figure 5: Dynamic monitoring of plasma Linc152 levels in NSCLC patients. (a) Correlation of Linc152 expression levels in tumor and plasma samples. (b) Comparison of plasma Linc152 levels between pre- and postoperative samples. (c) Scatter plots of plasma Linc152 levels from preoperative, postoperative, and recurrent patients, P <1. First, we confirmed the repeatability and precision of the qrt-pcr method by the intra-assay CV and interassay CV. Then, we investigated the expression and clinical significance of Linc152 in NSCLC. We found that Linc152 expression levels were higher in NSCLC tissues than in paired adjacent normal tissues and its levels correlated with tumor size. Our results were in accordance with the previous studies in other cancers [17, 18, 25], which suggested that Linc152 may play an oncogenic role in NSCLC. Circulating lncrnas were thought to be unstable due to the presence of RNase [27]. Thus, we next sought to analyze the stability of circulating Linc152. Our results suggested that Linc152 levels remained stable in plasma, which was consistent with the results in gastric cancer [15]. One possible explanation was that circulating lncrnas were packaged in exosomes to avoid the decomposition of RNase [25], and another possible explanation was that circulating lncrnas could be modified in some ways, including methylation, adenylation, and uridylation which make them resistant to RNase digestion [28]. In a word, these results demonstrated that Linc152 remained stable in plasma and whole blood, which could lay the foundation for evaluating plasma Linc152 as a biomarker for NSCLC patients. Then, we further found that plasma Linc152 levels were remarkably higher in NSCLC patients than in benign lung disease patients and healthy controls. Linc152 could provide a high diagnostic performance for distinguishing NSCLC patients from benign lung disease and healthy controls. Its AUC and sensitivity were higher than CEA, which is one of the most commonly used lung cancer biomarkers [29]. These results suggested that Linc152 could serve as a promising biomarker for NSCLC diagnosis. Moreover, the combination of Linc152 and CEA could provide a more accurate diagnosis than Linc152 or CEA alone. Henschke et al. reported the patients with early stage lung cancer who underwent resection within one month after diagnosis had a significantly improved 1-year survival rate [3]. Thus, it is important to diagnose NSCLC patients at early stage for improving the survival rate of NSCLC patients. We found that Linc152 could well distinguish NSCLC patients with early stage (AUC =.786, 95% CI: ) from healthy people, and the combination of Linc152 and CEA provided a more powerful diagnosis performance, which could substantially benefit to the treatment and prognosis of NSCLC. Finally, we carried out two analyses to validate whether plasma Linc152 levels could monitor NSCLC dynamics. Firstly, the correlation of Linc152 levels between plasma and tissue samples was analyzed. Plasma Linc152 levels had a significant association with tumor tissue. Secondly, Linc152 levels were analyzed in 47 paired pre- and postoperative plasma samples (after one month) from NSCLC patients. Plasma Linc152 levels were significantly decreased after operation. However, compared to NSCLC patients without recurrence, plasma Linc152 levels of patients with recurrence were sharply increased. The possible explanation was that circulating lncrnas were mainly derived from tumor cells [31], and it would presumably be back to normal after the removal of tumor, while recurrence of tumor will increase the supply to cause its abnormal raising. These results demonstrated that plasma Linc152 levels could reflect tumor dynamics and monitor the status of NSCLC patients after operation. In conclusion, this study investigated Linc152 in NSCLC patients for the first time. We found that Linc152 expression levels were upregulated in NSCLC tissues and correlated with tumor size. Moreover, we demonstrated that plasma Linc152 could serve as a promising biomarker for diagnosing and monitoring NSCLC patients. Conflicts of Interest The authors have declared that no competing interests exist. Authors Contributions Nandi Li and Xiao Bo Feng contributed equally to this work.

7 Disease Markers Acknowledgments This study was supported by the National Basic Research Program of China (973 Program) (212CB726, 212CB 7265) and the NSFC-NIH joint program on biomedical research ( ). References [1] T. Yang, B. Z. Chen, D. F. Li et al., Reduced NM23 protein level correlates with worse clinicopathologic features in colorectal cancers: a meta-analysis of pooled data, Medicine (Baltimore), vol. 95, article e2589, 216. [2] D. S. Ettinger, D. E. Wood, W. Akerley et al., Non-small cell lung cancer, version 6.215, the National Comprehensive Cancer Network, vol. 13, no. 5, pp , 215. [3] Y. Wu, H. Liu, X. Shi, Y. Yao, W. Yang, and Y. Song, The long non-coding RNA HNF1A-AS1 regulates proliferation and metastasis in lung adenocarcinoma, Oncotarget, vol. 6, no. 11, pp , 215. [4] T. Kishikawa, M. Otsuka, M. Ohno, T. Yoshikawa, A. Takata, and K. Koike, Circulating RNAs as new biomarkers for detecting pancreatic cancer, World Gastroenterology, vol. 21, no. 28, pp , 215. [5] H. 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